biostudies-literatureAlmaqwashi AANational Science FoundationNational Science Foundation Directorate for Biological Sciences667-676https://www.ebi.ac.uk/biostudies/studies/S-EPMC11900151biostudies-literatureUnknown124(4)Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA basepairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semirigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongatiBiophysical journalBinuclear ruthenium complex linker length tunes DNA threading intercalation kinetics.PMC11900151MCB-1817712Rouzina IAndersson JAlmaqwashi AAWesterlund FLincoln PMcCauley MJWilliams MCfalseBinuclear ruthenium complex linker length tunes DNA threading intercalation kinetics.Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA basepairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semirigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongati2025-01-01T00:00:00Z2025 Feb2026-07-16T05:07:02.062Z2026-07-10T03:08:33.881ZS-EPMC119001513979740310.1016/j.bpj.2025.01.002